•
• Asymmetrical membrane. Inside: negatively charged PLs; outside: neutral PLs; both sides: cholesterol (~30%).
• Both the PL composition and distribution, and the presence of proteins of influence on the membrane properties.
Cell membrane: bilayer of various phospholipids (PLs) with proteins ((ion) channels => transport of molecules across the membrane) [1].
DOPC/PE membranes
PC/PS membranes PI/PS membranes
Investigation of the influence of the composition of the membrane on its electrical properties ð Better understanding of the properties of the cellular membranes
ð Towards more controlled and reproducible cellular experiments.
BLMs = models for cell membranes
BLM preparation and charaterization Used (phospho)lipids
(found in natural cell membranes) Painting technique+ Easy
- Membrane reproducibility?
- Smaller membrane area
Montal-Mueller (MM) technique +
+ Asymmetrical membranes + Larger membrane area
- Tedious
Membrane reproducibility DOPC
PS PI
PE PC
CH
Iris van Uitert*, Yanina Cesa**, Hans de Boer*, Johan Bomer*, Martin Bennink**, Séverine Le Gac*, and Albert van den Berg*
* BIOS the Lab-on-a-Chip group, ** Biophysical Engineering, MESA+ Institute for Nanotechnnology, THE NETHERLANDS
E-mail: i.vanuitert@ewi.utwente.nl, Url: http://www.bios.ewi.utwente.nl
DOPC/PS membranes
DOPC/PI membranes
0 50 100 150 200 250 300 350
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1
X=DOPC/PI
Voltage(mV)
0 20 40 60 80 100 120 140 160 180
Capacitance(pF)
Breakthrough Voltage Capacitance
Cellular membrane
Bilayer lipid membrane
•
• Characterisation of membrane stability: measurement of the breakthrough voltage for many different membrane compositions, using binary membranes and membranes with more complex compositions mimicking the leaflets of biological cell membranes (MDCK cells).
• Membrane stability related to PL packing density and consequently to the number of insaturations in the hydrocarbon tails, the bulkiness of the headgroup and their charge.
• Insertion of a protein channel (áHL) in BLMs and first studies on the membrane stability.
Preparation of BLMs with mixtures of (phospho)lipids using the painting and MM techniques.
This work is funded by the POF group at the University of Twente.
[1] Alberts,
[2] Montal, M.; Mueller, P. Proceedings of the National Academy of Sciences of the United States of America 1972, 69, pp. 3561-3566.
[3] Krylov, A. V.; Pohl, P.; Zeidel, M. L.; Hill, W. G. Journal of General Physiology 2001, 118, pp.
333-339.
B. et al. (1989). Molecular biology of the cell. 2 ed.,New York: Garland Publishing Inc.
• Preparation of asymmetrical membranes using different (phospho)lipid compositions.
• Further electrical characterisation of BLMs that contain protein channels (á-HL, gramicidin).
• Study of the influence of the amount of inserted proteins on the stability of the membrane.
Influence of cholesterol in a membrane
0 50 100 150 200 250 300 350
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1
X=PC/PS
Voltage(mV)
0 20 40 60 80 100 120 140 160 180
Capacitance(pF)
Breakthrough Voltage Capacitance 0
50 100 150 200 250 300 350
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1
X=PI/PS
Voltage(mV)
0 20 40 60 80 100 120 140 160 180
Capacitance(pF)
Breakthrough Voltage Capacitance
Influence of the presence of proteins Pure lipid membranes
0 50 100 150 200 250 300 350
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1
X=DOPC/PS
Voltage(mV)
0 20 40 60 80 100 120 140 160 180
Capacitance(pF)
Breakthrough Voltage Capacitance
L- -Phosphatidylethanolamine (Heart, Bovine)a
L- -Phosphatidylinositol (Liver, Bovine-Sodium Salt)a L- -Phosphatidylcholine (Heart, Bovine)a
L- -Phosphatidylserine (Brain, Porcine-Sodium Salt)á 1,2-Diphytanoyl-sn-Glycero-3-Phosphocholine
0 50 100 150 200 250 300 350
0 2 4 6 8 10 12
% CH
Voltage(mV)
Breakthrough voltage
Properties of painted BLMs prepared from a mixture of 2 different PLs
Parameters influencing membrane stability
• Negative charge ð electrostatic repulsion
• Unsaturations in the hydrocarbon tails ð “kinking” of the tail
• Bulky headgroup
• PE and DOPC have the same headgroup.
• PE has one lipid tail with more unsaturations.
Linear of the and of
the of the membrane upon addition of PE (increased number of unsaturations) in the BLM due
to a in the .
decrease breakthrough voltage stability
decrease PL packing density
Decrease breakthrough voltage stability
decrease PL packing density
of the and of the
of the membrane upon addition of PI (addition of unsaturations, bulky headgroups and negative charges) in the BLM that causes a
in the .
Linear of the and
of the of the membrane upon addition of PI (more unsaturations, bulky headgroups and charges) in the BLM that causes a in the
.
decrease breakthrough voltage stability
decrease PL packing density
•
•
Lower packing density for 100% PS membranes.
Mixed membranes (50:50) than monoPL membrane: stabilizing charge interactions between the headgroups of DOPC and PS (ammonium NH moiety of DOPC with carboxylic 3+
moiety of PS) causing an in the .
more stable
increase PL packing density
Properties of BLMs prepared from more complex lipid mixtures Towards mimicking the membrane of cells ð
I n c r e a s e
breakthrough voltage stability
o f t h e
and of the of the membrane upon addition of cholesterol.
BLMs mimicking the inner/outer leaflets
•
• Outer leaflet than the inner leaflet (good agreement with results reported in the literature).
Mixed membranes more stable than monoPL membranes.
more stable
a-hemolysin (áHL) = channel protein.
• Rapid efflux of K and small molecules, influx of +
+ +
Na , Ca and molecules with MW < 1 kDa.2
• Pore-diameter: 1.5 nm.
• áHL used as a model of proteins found in cell membranes.
• Jumps in the current = insertion of pore proteins (1 jump = 1 protein inserted).
TOWARDS A BETTER UNDERSTANDING OF
BIOLOGICAL MEMBRANE PROPERTIES: INFLUENCE OF THEIR COMPOSITION ON THEIR STABILITY
BLMs created on a flat surface with a µhole
Cholesterol
DOPC: no unsaturation, neutral ð
PI: 4 unsaturations, charged, bulky head ð
highest Vbt ð most stable membrane
lowest Vbt ð least stable membrane
0 50 100 150 200 250 300 350
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1
X=DOPC/PE
Voltage(mV)
0 20 40 60 80 100 120 140 160 180
Capacitance(pF)
Breakthrough Voltage Capacitance
DOPC
• no unsaturation • neutral
PE
• 4 unsaturations
• neutral
DOPC
• no unsaturation
• neutral
PI
• 4 unsaturations
• negative charge
• bulky head
PI
• 4 unsaturations
• negative charge
• bulky head PS
• 1 unsaturation
• negative charge
DOPC
• no unsaturation
• neutral
PS
• 1 unsaturation
• negative charge
PS
• 1 unsaturation
• negative charge PC
• 2 unsaturations
• neutral
PL Unsaturations Charge Vbt(mV)
DOPC 0 x 204.00
PC 2 x 172.00
PS 1 168.33
PE 4 x 160.00
PI 4 91.67
GSL: Total Cerebrosides (Brain, Porcine). SM: Sphingomyelin (Egg, Chicken)
DOPC membranes; MM technique, insertion of áHL.
Stabilizing interactions between cholesterol and the hydrocarbon tails of the PLs [1].
350 µm aperture
PMMA substrate
PMMA substrate
PMMA substrate holder Device for BLM preparation
Device fitting on a microscope stage
PMMA Membrane
Introduction
Experimental
Results
Conclusion Outlook
Acknowledgements References
Hydrocarbon tail
Headgroup
Cis-double bond resulting in
•
•
Similar packing density for monoPL membrane: commpensation between the number of unsaturations and the charge.
Mixed membranes (50:50) than monoPL membrane (see above).
more stable
• Coupling electrical and optical
measurements using a home made set-up.
D e c r e a s e P L packing density
reduced stability i n t h e
resulting
in a of
the membrane.
Goal of this work
PLs (% in mix) PE (48.3%) PS (25.2%) PI (3.6%) CH (22.6%) Mix
Vbt(mV) 160.00 168.33 91.67 - 270.00
PLs (% in mix) GSL (26.8%) SM (23.8% ) PC (12.0% ) CH (37.3% ) Mix
Vbt(mV) - - 172.00 - 340.00
Inner leaflet
Outer leaflet
Painting of inner/outer mixtures of MDCK cells [3]
Painting of a CH- DOPC mixture
Stability of the membranes determined by measuring its breakthrough voltage (V )bt
